Removal of Adenine During a Pathogen Reduction Process in Whole Blood or Red Blood Cells by Dilution

ABSTRACT

The methods of this invention involve preventing the formation of a complex between adenine and riboflavin by reducing the amount of adenine in a solution containing blood or blood components to be pathogen reduced.

PRIORITY CLAIM

This application is a divisional of U.S. Non-provisional applicationSer. No. 10/423,200 filed Apr. 24, 2003, which claims priority from U.S.Provisional 60/375,849 filed Apr. 24, 2002.

BACKGROUND OF THE INVENTION

Contamination of blood supplies with infectious microorganisms such asHIV, hepatitis and other viruses and bacteria presents a serious healthhazard for those who must receive transfusions of whole blood oradministration of various blood components such as platelets, red cells,blood plasma, Factor VIII, plasminogen, fibronectin, anti-thrombin III,cryoprecipitate, human plasma protein fraction, albumin, immune serumglobulin, prothrombin, plasma growth hormones, and other componentsisolated from blood. Blood screening procedures which are currentlyavailable may miss contaminants. Thus, there is a need for sterilizationprocedures that effectively neutralize all infectious viruses and othermicroorganisms but do not damage cellular blood components, do notdegrade desired biological activities of proteins, and preferably do notneed to be removed prior to administration of the blood product to thepatient.

The use of photosensitizers, compounds which absorb light of a definedwavelength and transfer the absorbed energy to an energy acceptor, hasbeen proposed as a solution to the contamination of blood and bloodcomponents. Various photosensitizers have been proposed for use as bloodadditives for pathogen inactivation of blood or blood components. Areview of commonly used photosensitizers, and some of the issues ofimportance in choosing photosensitizers for decontamination of bloodproducts is provided in Goodrich, R. P., et al. (1997), “The Design andDevelopment of Selective, Photoactivated Drugs for Sterilization ofBlood Products,” Drugs of the Future 22:159-171.

Some photosensitizers that have been proposed for use for bloodcomponent photoirradiation have undesirable properties. For example,European Patent Application 196,515 published Oct. 8, 1986, suggests theuse of non-endogenous photosensitizers such as porphyrins, psoralens,acridine, toluidines, flavine (acriflavine hydrochloride), phenothiazinederivatives, and dyes such as neutral red and methylene blue, as bloodadditives. Another molecule, chlorpromazine, has been used as aphotosensitizer; however its usefulness is limited by the fact that itshould be removed from any fluid administered to a patient after thedecontamination procedure because it has a sedative effect.Protoporphyrin, which occurs naturally within the body, can bemetabolized to form a photosensitizer; however, its usefulness islimited in that it degrades the desired biological activities ofproteins.

Most preferred with respect to the reduction of pathogens in blood orblood products are endogenous photosensitizers. The term “endogenous”means naturally found in a human or mammalian body, either as a resultof synthesis by the body or because of ingestion as an essentialfoodstuff (e.g. vitamins) or formation of metabolites and/or byproductsin vivo. Examples of such endogenous photosensitizers are alloxazinessuch as 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin),7,8,10-trimethylisoalloxazine (lumiflavin), 7,8-dimethylalloxazine(lumichrome), isoalloxazine-adenine dinucleotide (flavine adeninedinucleotide [FAD]), alloxazine mononucleotide (also known as flavinemononucleotide [FMN] and riboflavine-5-phosphate), vitamin Ks, vitaminL, their metabolites and precursors, and napththoquinones, naphthalenes,naphthols and their derivatives having planar molecular conformations.The term “alloxazine” includes isoalloxazines.

The use of the endogenous alloxazine photosensitizers such as thosementioned above to reduce pathogens which may be contained in blood orblood products are disclosed in U.S. Pat. Nos. 6,258,577 and 6,277,337issued to Goodrich et. al and are herein incorporated by reference intheir entirety to the amount not inconsistent.

Endogenously-based derivative photosensitizers useful in this inventioninclude synthetically derived analogs and homologs of endogenousphotosensitizers which may have or lack lower (1-5) alkyl or halogensubstituents of the photosensitizers from which they are derived, andwhich preserve the function and substantial non-toxicity thereof. U.S.Pat. No. 6,268,120 to Platz et al. discloses alloxazine derivativeswhich may also be used to inactivate microorganisms contained in bloodor blood components. This patent is also incorporated by reference intothe present invention to the amount not inconsistent.

When certain endogenous photosynthesizers are used certain componentswhich are naturally occurring in blood plasma or in some synthetic bloodstorage/collection solutions may interact with the photosensitizerduring the photoinactivation process and form complexes. The presence ofthese complexes may increase the rate of side reactions which occurduring the photolysis of the photosensitizer. One such complex which mayform if 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin) is used asthe photosensitizer, is a complex between riboflavin and adenine.Adenine is found in blood plasma as well as being an additive componentof some synthetic blood collection/storage solutions.

It is toward this end of preventing damage to blood and blood componentsto be pathogen reduced by preventing the formation of aphotosensitizer-plasma constituent complex (such as adenine) that thepresent invention is directed.

Several U.S. Patents discuss the removal of plasma and plasma proteinsin a pathogen inactivation process using photosensitizers. U.S. Pat. No.5,360,734 issued Nov. 1, 1994 and U.S. Pat. No. 5,597,722 issued Jan.28, 1997 both to Chapman et al. discuss treating a blood componentcontaining red blood cells and plasma proteins by removing a portion ofthe plasma proteins before adding the photoactive agent benzoporphyrin.The treated blood component is prevented from contacting plasma proteinsfor a period of time (three to eighteen hours) after treatment toprevent binding of the treated cells to IgG proteins in the plasma.These patents do not disclose or suggest the removal of plasma toprevent the formation of specific plasma constituent-photosensitizercomplexes which changes the efficiency of the photosensitizer.

BRIEF SUMMARY OF THE INVENTION

Adenine is found naturally occurring in small concentrations in plasmaand in some synthetic blood collection/storage solutions. One method ofthis invention involves preventing the formation of a complex betweenadenine and riboflavin by reducing the amount of adenine in a solutioncontaining blood or blood components to be pathogen reduced by reducingthe level of plasma.

Another aspect of this invention involves the collection of blood orblood components to be pathogen reduced into pathogen reduction/storagesolutions which are adenine free.

If it is desired to pathogen reduce previously collected blood or bloodcomponents which were initially collected and stored in acollection/storage solution containing adenine, another aspect of thisinvention involves washing the previously collected blood componentswith saline or like solution, before the pathogen reduction process.

Another method which may be used for reducing the concentration ofselected components of plasma such as adenine in a fluid to be pathogenreduced may be by selective filtration.

After removal of adenine by any means known in the art, the fluidcontaining the blood component to be pathogen reduced is combined with aphotosensitizer such as riboflavin and exposed to photoradiation of theappropriate wavelength to activate the photosensitizer. The amount ofphotoradiation used is sufficient to activate the photosensitizer asdescribed herein, but less than that which would cause non-specificdamage to the biological components or substantially interfere withbiological activity of other proteins present in the fluid. Non-specificdamage is damage that damages all components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Jablonski diagram showing chemical reactions of7,8-dimethyl-10-ribityl isoalloxazine (riboflavin and other relatedcompounds) catalyzed by photoradiation, oxygen and other components.

FIG. 2 is a top plan view of a bag set containing a filter for removalof adenine for use in a pathogen reduction procedure.

FIG. 3 shows an embodiment of this invention using a bag to contain thefluid being treated with the photosensitizer and a shaker table toagitate the fluid while exposing to photoradiation from a light source.

FIG. 4 is a graph comparing the % hemolysis of pathogen reduced redblood cells stored over time in pathogen reduction/storage solutionswith and without adenine.

DETAILED DESCRIPTION OF THE INVENTION

The pathogen reduction method of this invention using endogenousphotosensitizers and endogenously-based derivative photosensitizers isexemplified herein using 7,8-dimethyl-10-ribityl isoalloxazine as thephotosensitizer.

7,8-dimethyl-10-ribityl isoalloxazine (riboflavin or vitamin B2) absorbslight from about 200 to 500 nm. The ring system core of7,8-dimethyl-10-ribityl isoalloxazine is resistant to photodegradationbut the ribityl side chain of riboflavin undergoes photodegradation.

Photosensitizers of this invention include compounds whichpreferentially adsorb to nucleic acids, thus focusing their photodynamiceffect upon the nucleic acids of microorganisms and viruses with littleor no effect upon accompanying cells or proteins. Pathogen kill usingriboflavin and related compounds also occurs upon photoinactivation viasinglet oxygen damage, thereby disrupting the ability of the pathogen tofunction and reproduce or both.

FIG. 1 is a Jablonski diagram showing the photochemical reactions of7,8-dimethyl-10-ribityl isoalloxazine (riboflavin and other relatedcompounds) which occur upon catalysis by photoradiation, oxygen andother components. The photosensitizer in its ground state is referred toas S₀. Upon absorption of light, riboflavin is converted to anelectrically excited state which in condensed phase immediately(<<10⁻¹¹s) relaxes to the lowest vibrational level of the lowest excitedstate (S₀). The lifetimes of S₁ states in solution are usually in therange of 1-10 ns and are controlled by internal conversion (IC) andfluorescence (F) decay back to S₀ by intersystem crossing (ISC) to aparamagnetic triplet state (T₁) and by inter and intramolecular chemicalreactions. As is known in the art, internal conversion is theradiationless transition between energy states of the same spin state.Intersystem crossing (ISC) is a radiationless transition betweendifferent spin states. When the riboflavin relaxes from the singletstate to the ground state, it is called fluorescence. When the moleculerelaxes from the triplet state (S₁) to the ground (unexcited) state (S₀)this is called phosphorescence.

The left arrow (first vertical, upward-pointing arrow) in the diagram ofFIG. 1 indicates that upon absorption of light energy the riboflavinmolecule can go from its ground state (S₀) to its excited sate (S₁) andbecome involved in chemical reactions including losing its ribitylmoiety to become lumichrome (7,8-dimethylalloxazine). Lumichrome is notphotoactive under visible light.

Alternatively, as shown by the second vertical, downward pointing arrowthe excited molecule may release its absorbed energy and fluoresce toreturn to the ground state. The wavy arrows indicate that energy isreleased.

The excited riboflavin molecule may also relax to its triplet state (T₁)through intersystem crossing (ISC) by changing the spin of an electron(spin conversion). The wavy line labeled ISC indicates intersystemcrossing. If no oxygen is present, the molecule in its triplet state canphosphoresce (second wavy, downward pointing arrow) and return to itsground state. Or, as indicated by the right arrow, the molecule in itstriplet state can react with other molecules in close proximity andreturn to its ground state. IF oxygen is present, the molecule in itstriplet state can react with oxygen and return to its ground stateproducing ¹O₂ (singlet oxygen). Singlet oxygen can cause DNA strandbreaks, further contributing to pathogen kill.

One disadvantage of using the described photochemical methods forpathogen reduction of blood products is that the singlet oxygen speciesgenerated in the process of photolysis of riboflavin may cause damage toblood products and compromise their suitability for transfusions. Ifcertain plasma proteins or other components of plasma are present duringthe photolytic process, the presence of such components may magnify thisoxidative process.

One component found in blood plasma and in some commonly used bloodstorage solutions which, if present, has been suggested to have aneffect on the oxidative process of riboflavin, is the nucleosideadenine. Uchara et al. has shown that upon photoactivation, a specificcomplex is formed between riboflavin and adenine which increases thephotodynamic efficiency of riboflavin. The authors showed anaccelerative effect of the riboflavin-adenine complex on thephotodynamic inactivation of yeast alcohol dehydrogenase. (KinachinoUehara, Tadashi Mizoguchi, Morio Yonezawa, Saburo Hosomi and RyogiHayashi, Effect of Adenine on the Riboflavin-sensitized Photoreaction 1.Effect of Adenine on the Photodynamic Inactivation of Yeast AlcoholDehydrogenase in the Presence of Ribflavin, J. Vitaminology 17, 148-154(1971.))

While the formation of a riboflavin-adenine complex may appear to be adesirable side effect in that the presence of the complex would help todecrease the time necessary to pathogen reduce any pathogens containedin and/or around blood or blood components, in fact, the presence of thecomplex speeds up the oxidative chemistry of riboflavin. The increase inproduction of reactive oxygen species produced during the oxidation ofriboflavin, increases the possibility of cell membrane damage. Cellswhich are damaged during a pathogen reduction procedure are unable to bereinfused into a patient.

Because adenine is naturally occurring in plasma, in one embodiment ofthe present invention, the adenine content of fluid to be pathogenreduced is reduced by reducing the plasma content. One method suitablefor the plasma reduction step is to dilute the fluid containing plasmawith an adenine-free diluting solution. This will reduce the level ofadenine in the fluid to be pathogen reduced, thus reducing the amount ofadenine available to form a complex with riboflavin. The dilutingsolution used to reduce the level of adenine to an amount which will notform a complex with riboflavin may be one of many different solutions,including saline; a physiologic buffer, which may comprise a variety ofdifferent substances; a solution containing glucose, phosphate or both,which may or may not act as a buffer; a solution containing nutrients; acryopreservative: an anticoagulant; a cell storage solution known to theart or developed to provide cells with suitable additives to enable themto be stored or infused; or other suitable solution.

The diluting solution should not substantially interfere with theinactivation of microorganisms or substantially destroy the biologicalactivity of the fluid. By “substantially interfere” is meantinterference which is sufficient to prevent pathogen reduction fromoccurring at a desired level.

The diluting solution may also contain a substrate which selectivelybinds to adenine, effectively removing it from the fluid by rendering itunable to bind to riboflavin. Although in this method adenine may stillbe present in the fluid to be pathogen reduced, the adenine which ispresent is not available to bind to riboflavin because it is bound tothe adenine-binding substrate. One such adenine-binding substrate whichmight be used in this invention may be an antibody directed againstadenine. The antibody could be added directly to the adenine-containingsolution to be pathogen reduced, or could be coupled to a substrate suchas polymeric beads. Another substrate which may be used to removeadenine from the fluid may be an ion exchange resin. Such a resin wouldpreferentially bind to adenine based upon the ionic charge of adenine,thus effectively removing adenine from the fluid.

Another method which may be used for reducing the concentration ofselected components of plasma such as adenine in a fluid to be pathogenreduced may be by selective filtration. Such methods of filtering outunwanted substances such as adenine from fluids are known in the art.One example of a filter which may be used to selectively remove adenineis a hollow fiber filter. The pore sizes of this filter would be smallenough to allow adenine to pass through the pores and be removed fromthe fluid, leaving the blood component to be pathogen reduced behind.

Another method of selectively filtering out adenine which may be usefulwith the present invention is to use a filter having an absorptionligand on its surface which selectively binds to adenine, thuseffectively removing adenine from the fluid to be pathogen reduced. Thismethod would allows the plasma (minus adenine) to be retained as part ofthe fluid to be pathogen reduced.

FIG. 2 depicts one example of a bag set for use in a pathogen reductionprocedure containing a filter which may be used to remove adenine fromthe fluid to be pathogen reduced. Fluid containing blood and plasma, ora collected blood component which has been previously collected in acollection storage solution containing adenine is contained in bag 10.To substantially remove all adenine which may be contained therein, thefluid to be pathogen reduced flows out of bag 10 via exit port 2 throughtubing 7 and into filter 5. Filter 5 may contain filter media having asubstrate thereon which selectively binds to adenine, thus removing itfrom the fluid. After flowing through the filter, the now substantiallyadenine-free fluid flows through tubing 9 and into bag 12 via port 4.Bag 12 may be prepackaged to contain riboflavin, or riboflavin may beadded after the now adenine-free fluid to be pathogen reduced is flowedinto bag 12.

In another embodiment, the adenine removal filter may also be containedwithin one of the bags 10 or 12. The adenine contained in the fluidwould bind directly to the filter contained within the bag, and transferof the now adenine-free fluid into another bag would not be needed.

The adenine reducing step may also be carried out using mechanical meanssuch as centrifugation, to separate the fluid containing adenine fromthe blood component to be pathogen reduced. This centrifugation step maybe done using an apheresis machine such as the COBE Spectra™ or TRIMA®apheresis systems available from Gambro BCT Inc. (Lakewood, Colo., USA)as well as apheresis systems of other manufacturers. The separated bloodcomponents may then be resuspended in a suitable solution which does notcontain adenine. The reduction step may also comprise washing theseparated blood component to be pathogen reduced one or more times, asis known in the art. One machine suitable for washing the blood orseparated blood components is the COBE 2991 (also available from GambroBCT Inc.) Washing is generally the addition to the blood component to bepathogen reduced a solution which does not contain adenine to dilute thepercentage of plasma (or of collection/storage solution) aidconsequently the amount of adenine. The wash solution is removed and apathogen reduction solution may be added to resuspend the washedcomponents. The process may be carried out one or more times dependingon the initial level of adenine contained in the fluid.

The fluid to be pathogen inactivated may also be initially collectedinto a solution which does not contain adenine. If this is the case, nostep of removing adenine is needed.

In a batch-wise process, after substantially removing any adenineinitially present in the plasma or in the collection/storage solution,the fluid to be pathogen reduced is placed into bags which arephotopermeable or at least sufficiently photopermeable to allowsufficient radiation to reach their contents to activate thephotosensitizer. Photosensitizer is added to each bag to substantiallyinactivate any pathogens which may be contained therein, and the bag ispreferably agitated while irradiating, for a period of time to ensureexposure of substantially all the fluid to radiation.

FIG. 3 depicts an embodiment of this invention in which fluid to bedecontaminated and which is substantially adenine-free is placed in abag 284 equipped with an inlet port 282, through which photosensitizer290 may be added from flask 286 via pour spout 288. Shaker table 280 isactivated to agitate the bag 284 to mix the fluid to be decontaminatedand the photosensitizer together while photoradiation source 260 isactivated to irradiate the fluid and photosensitizer in bag 284.Alternatively, the bag can be prepackaged to contain photosensitizer andthe fluid to be pathogen reduced is thereafter added to the bag.

It is also contemplated that the pathogen reduction process can be donein a flow-through system. In a flow-through process, after substantiallyremoving any adenine initially present in the plasma or in thecollection/storage solution, a photosensitizer is added to the fluidcontaining a blood component which is to be pathogen reduced. Thephotosensitizer and blood component is flowed past a photoradiationsource, and the flow of the material generally provides sufficientturbulance to distribute the photosensitizer throughout the fluid. Amixing step may optionally be added.

EXAMPLES

Blood to be pathogen reduced may be separated into components by anymean is known in the art.

Example 1

The method of this example requires the removal of substantially alladenine which may be contained in a solution used to resuspend and/orcollect platelets to be pathogen reduced. Removal of adenine may be doneusing any of the methods set forth above. If an adenine-free solution isused to resuspend or collect the platelets to be pathogen reduced, noadenine removal step is needed. After removal of any adenine which maybe present, the photosensitizer is mixed with the fluid containingplatelets. Mixing may be done by simply adding the photosensitizer or asolution containing the photosensitizer to the platelets to be pathogenreduced. In one embodiment, the material to be decontaminated to which aphotosensitizer has been added is flowed past a photoradiation source,and the flow of the material generally provides sufficient turbulence todistribute the photosensitizer throughout the fluid to be pathogenreduced. A nixing step may optionally be added. In another embodiment,the fluid and photosensitizer are placed in a photopermeable containerand irradiated in batch mode (see FIG. 2), preferably while agitatingthe container to fully distribute the photosensitizer and expose all thefluid to the radiation.

The amount of photosensitizer to be mixed with the fluid to be pathogenreduced will be an amount sufficient to adequately inactivate thereproductive ability of a pathogen. Preferably the photosensitizer isused in a concentration of at least about 1 μM up to the solubility ofthe photosensitizer in the fluid. For 7,8-dimethyl-10-ribitylisoalloxazine a concentration range between about 1 μM and about 160 μMis preferred, preferably about 50 μM.

The wavelength used will depend on the photosensitizer selected, and thetype of blood component to be pathogen reduced. For platelets andplasma, a light source is used which provides light in the range ofabout 200 nm to about 320 nm, and more preferably about 308 nm may beused. For red blood cells, light in the range of about 200 nm to about600 nm is used, preferably about 447 nm.

The following storage solutions shown in Table 1a and 1b are examples ofcommonly used platelet storage solutions which may be used with thisinvention. These solutions may be used to resuspend platelets to bepathogen reduced before the addition of the photosensitizer, or may beused to resuspend platelets after a pathogen reduction procedure. Othersolutions not specifically listed that do not contain adenine may alsobe used. It should be noted that platelets may also be resuspended inbuffer and/or saline as long as no adenine is present. TABLE 1a PAS IIPSM1-pH PlasmaLyte A Molecular Conc. g/300 Conc. g300 Conc. g/300 Weight(mMol/L) mLs (mMol/L) mLs (mMol/L) mLs Sodium Chloride 58.44 115.5 2.0298 1.72 90 1.58 Potassium 74.55 0.00 5 0.11 5 0.11 Chloride CalciumChloride 111 0.00 0.00 0.00 Magnesium 95.21 0.00 0.00 3 0.09 ChlorideMagnesium 120.4 0.00 0.00 0.00 Sulfate Tri-Sodium 294.1 10 0.88 23 2.0323 2.03 Citrate Citric Acid 192.1 0.00 0.00 0.00 Sodium 84.01 0.00 0.000.00 Bicarbonate Sodium 142 0.00 25 1.07 0.00 Phosphate Sodium Acetate82.03 30 0.74 0.00 27 0.66 Sodium 218.1 0.00 0.00 23 1.50 GluconateGlucose 180.2 0.00 0.00 0.00 Maltose 360.3 0.00 0.00 0.00 Adenine 135.10.00 0.00 0.00Note:Assumes that all salts are anhydrous

TABLE 1b SetoSol PAS III PAS Molecular Conc. g/300 Conc. g/300 Conc.g/300 Weight (mMol/L) mLs (mMol/L) mLs (mMol/L) mLs Sodium Chloride58.44 90 1.58 77 1.35 110 1.93 Potassium 74.55 5 0.11 0.00 5.1 0.11Chloride Calcium Chloride 111 0.00 0.00 1.7 0.06 Magnesium 95.21 3 0.090.00 0.00 Chloride Magnesium 120.4 0.00 0.00 0.8 0.03 Sulfate Tri-Sodium294.1 17 1.50 12.3 1.09 15.2 1.34 Citrate Citric Acid 192.1 0.00 0.002.7 0.16 Sodium 84.01 0.00 0.00 35 0.88 Bicarbonate Sodium 142 25 1.0728 1.19 2.1 0.09 Phosphate Sodium Acetate 82.03 23 0.57 42 1.03 0.00Sodium 218.1 0.00 0.00 0.00 Gluconate Glucose 180.2 23.5 1.27 0.00 38.52.08 Maltose 360.3 28.8 3.11 0.00 0.00 Adenine 135.1 0.00 0.00 0.00Note:Assumes that all salts are anhydrous

Example 2

Example 2 is directed toward the removal of adenine in a fluidcontaining red blood cells to be pathogen reduced. If ariboflavin-adenine complex forms in a solution containing red bloodcells, the increased oxidation reactions caused by the presence of thecomplex may damage the red blood cell membranes, causing hemolysis andincreased methemoglobin formation. Methemoglobin formation isundesirable because methemoglobin does not allow the red blood cells toefficiently bind and deliver oxygen.

This phenomenon is shown in FIG. 4, which shows the % hemolysis of redblood cells over time in solutions with and without adenine. Red bloodcells were suspended in AS3 during a pathogen reduction procedure usingriboflavin and visible light. AS3 is an AABB approved red blood cellpreservative. AS3 contains sodium chloride, dextrose, adenine, sodiumphosphate, sodium citrate and citric acid. As can be seen in FIG. 4, redblood cells suspended in 5% AS3 show the highest percentage of red bloodcell hemolysis. Red blood cells subjected to a pathogen reductionprocedure in a solution containing no adenine (0% AS3) show less than 2%hemolysis of red blood cells.

Red blood cells to be pathogen reduced should be collected in ananticoagulant-preservation solution which does not contain adenine.Other anticoagulant-preservation solutions not specifically listed inTable 2a and 2b below that do not contain adenine may also be used. Ascan be seen from Table 2a and 2b, none of the anticoagulant-preservativesolutions listed below contain additional adenine. TABLE 2aANTICOAGULANT PRESERVATIVE SOLUTIONS CPD CP2D Molecular Conc. Conc.Weight (mMol/L) mg/63 ml mg/100 ml (mMol/L) mg/63 ml mg/100 ml SodiumCitrate 294.1 89.59 1660.00 2634.92 89.59 1660.00 2634.92 Citric Acid192.1 15.53 188.00 298.41 15.53 188.00 298.41 Dextrose 180.2 141.821610.00 2555.56 283.64 3220.00 5111.11 Monobasic 120 18.52 140.00 222.2218.52 140.00 222.22 Sodium phosphate Adenine 135.1 0.00 0.00 0.00 0.000.00 0.00

TABLE 2b ANTICOAGULANT PRESERVATIVE SOLUTIONS ACD-A ACD-B MolecularConc. Conc. Weight (mMol/L) mg/100 ml (mMol/L) mg/100 ml Dextrose 180.2135.96 2450.00 81.58 1470.00 Adenine 135.1 0.00 0.00 0.00 0.00 Monobasic120 0.00 0.00 0.00 0.00 sodium phosphate Mannitol 182.2 0.00 0.00 0.000.00 Sodium 58.45 0.00 0.00 0.00 0.00 Chloride Sodium 294.1 74.802200.00 44.88 1320.00 Citrate Citric Acid 192.1 41.64 800.00 24.99480.00

Alternatively, if previously collected red blood cells are to bepathogen reduced, the cells may be washed before undergoing a pathogenreduction procedure to remove any adenine contained in the solution usedto collect and store the previously collected cells. The washingprocedure may be used to remove plasma (which contains endogenousadenine), or to remove adenine from blood products which were previouslycollected and stored in synthetic storage solutions or anticoagulantscontaining exogenous adenine.

One red blood cell wash process which may be used with the presentinvention is described below. However, any process for washing cellsknown in the art may be used. Red cells can be washed by manualcentrifugation or with an automated cell washer such as the COBE 2991(available from Gambro BCT, Lakewood, Colo., USA). The 2991 washes thered cells with 700 mL of 0.9% sodium chloride and 300 mL of 500 μMriboflavin in 0.9% sodium chloride.

The product of the wash step is a suspension of concentrated red bloodcells at a 60 to 70% hematocrit. The washed red cells are mixed with asolution containing 550 μM riboflavin in a 0.9% sodium chloride toobtain a suspension with a hematocrit of 50% and a volume of 276 mL. Thesolution may also be any of the anticoagulant-preservative solutions setforth in the tables above.

The washed red cells are transferred from the cell-washing bag to a bagsuitable for illumination and subsequent dilution to a 50% hematocrit.The washed red cells and riboflavin arc typically illuminated withvisible light at a wavelength of 447 nm and 120 J/cm2. Afterillumination, the extracellular fluid is expressed off and a storagesolution which may or may not contain adenine is added in an amountnecessary to increase the hematocrit of the red cells to 55%. Thepathogen reduced red blood cells may then be stored or directlyreinfused into a patient.

Removal of adenine may also be done using any of the other methods setforth above.

The addition of “quenchers” or oxygen scavengers, may be used to enhancethe pathogen reduction process by further reducing the extent ofnon-specific cell-damaging chemistry. Examples of quenchers which may beused in this invention include electron rich amino acids such ashistidine, methionine, tyrosine and tryptophan. Nucleotides such ascysteine, guanosine and adenoside monophosphate. Sulfhidryl quencherssuch as N-acetyl-L-cysteine and glutathione. Antioxdants such as trolox,Vitamin E and alpha-tocopherol acetate. Other quenchers such as propylgallate, ascorbate, mercaptopropionylglycine, dithiothreotol,nicotinamide, BHT, BHA, lysine, serine, glucose, mannitol, glycerol, andmixtures thereof may also be used. Quenchers may be added to the fluidto be pathogen reduced either before or after the removal of adenine.

It will be appreciated that the instant specification and claims are setforth by way of illustration and not of limitation, and that variousmodifications and changes may be made without departing from the spiritand scope of the present invention.

1. A method for treating a selected quantity of fluid to reduce pathogens which may be present therein, the fluid containing one or more components selected from the group consisting of blood and blood components, the method comprising: (a) adding a sufficient volume of a washing solution to the selected quantity of fluid; (b) washing the selected quantity of fluid to create a washed fluid; (c) mixing a pathogen reduction-effective, substantially non-toxic amount of 7,8-dimethyl-10-ribityl isoalloxazine photosensitizer with the washed fluid; (d) exposing the washed fluid containing the photosensitizer to photoradiation of sufficient wavelength and energy to activate the photosensitizer, whereby the pathogens are reduced.
 2. The method of claim 1 wherein the washing step further comprises a plurality of washing steps.
 3. The method of claim 1 wherein the method substantially prevents damage to the blood or blood components contained in the fluid by reducing the concentration of adenine in the fluid to prevent formation of a photosensitizer-adenine complex.
 4. The method of claim 1 wherein the method substantially prevents an increase in oxidation reactions produced by photolysis of the photosensitizer.
 5. The method of claim 1 wherein the mixing step further comprises placing the fluid in a container transparent to photoradiation, adding the photosensitizer to the fluid in the container and agitating the container.
 6. The method of claim 1 wherein the washed fluid consists essentially of platelets.
 7. The method of claim 1 wherein the washed fluid consists essentially of red blood cells.
 8. A fluid for pathogen reduction that is substantially adenine-free comprising: one or more components selected from the group consisting of blood and blood components; an endogenous photosensitizer or an endogenously-based derivative photosensitizer and an adenine-free solution. 